1 .Field of the Invention
The invention relates to new cultures of keratinocytes.
The invention also relates to a process for preparing the same.
The invention also relates to the use of new cultures of keratinocytes as wound healing substances.
The invention also relates to pharmaceutical compositions containing as active substances, said new cultures of keratinocytes.
The invention also relates to cosmetic compositions, containing as active substances, new cultures of keratinocytes.
2. Description of the Prior Art
Skin is presumably the organ most subject to injury. Skin repair is a complex process that can be divided in 4 phases usually described as inflammation, granulation tissue formation, epithelialization and remodeling of the connective tissue matrix. Each of these phases is complex in itself, and it is clear that for good wound healing, the processes must occur successively and in coordination. Good wound healing can be defined as restoration of the skin, including the dermal and epidermal part, in such a way that the resulting scar tissue maximally resembles the unwounded skin structurally, histologically, functionally, and esthetically. Obviously, such scar tissue is different from a hypertrophic scar or keloid.
For purposes of clarity a simplified description of the composition of human skin is given below. The upper part is composed of the epidermis, which contains mostly keratinocyte or epithelial cells, some melanocytes and Langerhans cells, and several Merkel cells. Five different layers are found in the epidermis, reflecting the state of keratinization. The proliferating keratinocytes at the base of the epidermis, i.e., in the stratum basale, are attached to the dermis via the basement membrane. The dermis is composed of connective tissue, including fibroblasts and other connective tissue cells, and connective tissue matrix substances. Blood vessels, nerves, sensory organs, sweat glands, sebaceous glands, and hair follicles are present in the dermis.
Clinical and animal experiments have demonstrated that application of in vitro cultured (human) keratinocytes, for instance as sheets, induces wound healing in chronic wounds such as ulcers and in burns, which may be treated concomitantly with meshed split skin autografts. Moreover, there are indications that the application of cultured keratinocyte grafts suppresses hypertrophic scar formation and keloid formation. Initially, autografts were used, which were prepared by growing keratinocytes isolated from the patients own skin. Confluent (differentiated) keratinocyte cultures are then detached from the culture dish and applied as a sheet, with basal cells facing downwards on the wound. About 3 weeks are needed before a reasonable amount of keratinocytes can be cultured for application as an autograft. Even then, the amount may not be sufficient to cover the entire wound surface. However, this time period may be critical for the patient, especially in the case of extensive third degree burns.
Therefore, experiments were undertaken to apply keratinocyte allografts. Keratinocytes are isolated from the skin of one person, cultured to obtain confluent keratinocyte sheets, and then applied on the wound of a patient. No differences in wound healing activity were observed between auto- and allografts. A main disadvantage of cultured allografts is the risk of transferring pathogens such as human immunodeficiency virus, hepatitis B virus, and cytomegalovirus, from the skin donor to the acceptor patient.
These techniques for wound treatment make use of fresh keratinocyte grafts, containing proliferating keratinocytes. The keratinocyte sheets are detached from the culture vessel by treatment with Dispase and immediately applied onto the wound. The availability of these sheets in terms of time and quantity is a major drawback of fresh keratinocyte grafts. It is assumed that proliferating keratinocyte cultures contain higher wound healing activity than more differentiated cultures. Moreover, only a multilayer culture can be detached as a sheet from the culture vessel, and can be spread over the wound. Therefore, cultured keratinocytes have to be taken at an optimal growth phase. However, it is impossible to predict when a patient will enter the hospital and how many keratinocyte grafts will then be needed. Another drawback of using fresh (autologous) keratinocyte sheets is that a certain period of time is still required before a sufficient amount of keratinocyte sheets can be grown and then applied.
Cryopreserved allografts partially circumvent these inconveniences. Cryopreserved keratinocyte grafts can be prepared as follows: confluent (differentiated) keratinocyte cultures are rinsed with phosphate buffered saline (PBS), cultured for 1 day without additives (epidermal growth factor, insulin, cholera toxin, triiodothyronin, transferrin, hydrocortisone), detached from the dish by treatment with Dispase and adhered to a transfer substrate (e.g., Interface.RTM. Wuhrlin-Soplamed). The sheets are soaked in culture medium supplemented with 10% DMSO as cryoprotectant, and stored frozen in liquid nitrogen or for a shorter period of time at -80.degree. C. Before application, the specimen is thawed and rinsed with PBS in order to remove DMSO and bovine serum proteins. The sheets are applied with basal cells facing downwards as for fresh sheets. After thawing, about 60% of the cells are viable as judged from vital staining.
Initially, it was assumed that the keratinocytes from cultured allografts remain and grow on the wounds, an event which is clinically known as "take". However, several reports recently demonstrated that wound closure is due to growth of host epithelium.
Thus, healing is induced without permanent take of keratinocyte grafts, suggesting that the wound healing activity may be due to a (chemical) substance(s) associated with the cultured keratinocytes, which stimulates keratinocyte outgrowth. Consequently, it would not be necessary to apply sheets of viable keratinocytes. Therefore, the cultured keratinocyte sheets were lyophilized and the wound healing activity was compared with fresh and cryopreserved sheets.
Hence, the beneficial effect of cultured keratinocyte sheets in wound healing can probably be attributed to (a) substance(s) present in the cultured keratinocyte or induced by (a) molecule(s) present in the cultured keratinocytes. To date the(se) factor(s) and their concentration in the cultured keratinocytes are unknown. It cannot be excluded that certain known cytokines and growth factors are present. Many known growth factors, e.g., fibroblast growth factor (FGF), platelet derived growth factor (PDGF), epidermal growth factor (EGF), transforming growth factor alpha (TGF-.alpha.), transforming growth factor beta (TGF-.beta.) influence keratinocytes and skin fibroblasts. For instance, TGF-.alpha., EGF, and FGF induce keratinocyte proliferation. TGF-.beta. and PDGF stimulate the synthesis of collagen and other connective tissue components. Neovascularisation can be influenced by basic-FGF and TGF-.alpha.. Because of these properties, such growth factors may play a role in wound healing.
Clinical results showed that fresh and cryopreserved cultured keratinocyte sheets stimulate re-epithelialization, resulting in quicker healing compared to classic wound treatment or application of meshed split skin autografts only. According to the results of our animal model for wound healing, lyophilized cultured keratinocytes gave better results than keratinocyte sheets.
The term "lyophilized keratinocytes" denotes the product obtained from human or animal keratinocytes, which have been grown in vitro to the subconfluent, confluent, or differentiated state, and then a lyophilisate is prepared of these cells in such a way that no or minimal degradation of cell-associated substances occurs. Such a lyophilisate contains wound healing activity. Cell extracts from these cultured keratinocytes also contain wound healing activity. A cell extract may be prepared, for instance, after lysis and/or disruption of the cultured keratinocytes and subsequent fractionation. The total material or separate fractions may be lyophilized. Lyophilization is not necessary, but it is advantageous in that the resulting substance is easier to store, can be kept in a small place, is easier to handle, can be applied in a formulation (e.g., in a dry powder, an ointment, a suspension, a solution, a gel, a creme or a biocompatible, synthetic or natural, solid matrix) chosen according to the circumstances, can be administered at an optimal dose, normally has a longer shelf life, and can easily be screened for the most active preparation.
One advantage of lyophilized cultured keratinocytes is that the material is readily available exactly at the moment it is needed, in contrast to fresh cultured keratinocyte sheets. Before application on a wound, fresh keratinocyte sheets have to be cultured for one day without fetal calf serum and additives such as epidermal growth factor, cholera toxin, triiodothyronin, transferrin, etc. Subsequently, the sheets have to be removed from the culture dish with Dispase, rinsed and transferred to the operation room in a sterile manner. In the case of cryopreserved sheets, the storage medium, containing 10% DMSO as cryoprotectant, is thawed as quickly as possible at 37.degree. C. and thoroughly rinsed with PBS before it is transferred to the operating room. Thawed cryopreserved keratinocyte grafts have to be handled carefully since the sheets are extremely fragile. Besides the need for specialized accommodations, it takes at least 1 day for fresh keratinocyte grafts and about 1 h in the case of cryopreserved keratinocyte grafts, before they can be applied on the wound. In contrast, lyophilized keratinocyte material is instantly available and may be applied as such, or rehydrated in previously prepared sterile gel or salt solution. Lyophilized-keratinocyte derived substances may be applied at the optimal dose. In the case of keratinocyte sheets, the wound is covered with a sheet and it is assumed to be useless to put another sheet on top.
Interdonor differences play a role in the activity of the cultured keratinocytes. The keratinocyte proliferation stimulating activity of lyophilized keratinocyte-derived substances is easily tested in vitro. The most active batches can be selected for patient treatment. Fresh keratinocyte grafts cannot be tested and, in the case of cryopreserved keratinocyte sheets, the preparation of the material requires additional work.
Lyophilized cultured keratinocytes, extracts or fractions are easy to store. A large amount may be prepared beforehand. Another advantage is that keratinocyte growth stimulatory activity may be tested in vitro in order to select the most active preparations. This is obviously impossible when fresh keratinocyte sheets are used. Moreover, fresh sheets must be used at an optimal time and may not be stored at all. If there are no patients to treat at that optimal moment of culture, the cells become worthless for this purpose. Besides, cryopreserved keratinocyte sheets should be stored in a cryobiological storage vessel containing liquid nitrogen. The stock of lyophilized keratinocytes can be kept at -20.degree. C. as dry powder.
Lyophilized keratinocytes do not require special transportation facilities. By contrast, when detached from the culture surface, fresh and cryopreserved keratinocyte grafts have to be transported in isotonic, sterile buffered medium to prevent drying of the keratinocytes. Moreover, these keratinocyte grafts may detach from the supporting gauze, and start to float or curl up, hampering the exact method of application, i.e., basal keratinocytes facing downwards. Lyophilized keratinocyte material is a light weight powder, simply kept in a sterile vial. It is advised to rehydrate and/or redissolve the powder in gel or saline prior to use. The preparation can then be kept for at least 1 week at 4.degree. C. without loss of keratinocyte proliferation stimulating activity. Rehydrated substance may have better contact with the wounded tissue, thereby favoring the healing process.
Since the substance is completely dry during storage, proteinases are not active, thus increasing the shelf life.
Some cultures of keratinocytes which are used either for skin grafting, wound healing or for fundamental studies require cultures containing fibroblasts, such as 3T3.
For instance, the document titled "Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells" (J. G. Rheinwald et al., Cell, vol. 6, pp. 331-344, Nov. 1975) describes the presence of fibroblasts to initiate colony formation of human epidermal keratinocytes, but states that proliferation of fibroblasts must be controlled so that the epidermal cell population is not overgrown. Both conditions can be achieved by the use of lethally irradiated 3T3 cells at the correct density.
Another example where 3T3 cells are used is the culture of keratinocytes described in "Growth of cultured human epidermal cells into multiple epithelia suitable for grafting" (H. Green et al., Proc. Natl. Acad. Sci. USA, vol. 76, n. 11, pp. 5665-5668, November 1979).
In order to avoid the use of neoplastic cells, i.e., the 3T3 fibroblasts, in cultures for application on open wounds, several reports have described feeder layer-free keratinocyte culture techniques. Feeder layer-free keratinocyte cultures have been performed on special substrates (e.g., on fibronectin (Gilchrest B. A. J. Cellular Physiology, 1982) or collagen-coated substrates (Hawley-Nelson P. J. Invest. Dermatol., 1980)) or the cells were plated at higher densities on an uncoated surface of culture plastic (Eisinger M. PNAS 1979).
Some authors have proposed to cultivate keratinocytes on media which are free from fibroblasts, but which contain bovine pituitary extracts or bovine brain extracts.
More precisely, Mark R. Pitellkow et al. have disclosed in "New techniques for the in vitro culture of human skin keratinocytes and perspectives on their use for grafting of patients with extensive burns" (Mayo Clinic Proceedings, vol. 61, pp. 771-777, 1986) a culture method involving a two-phase technique: proliferation (phase 1) and differentiation (phase 2). Phase 1 is performed in medium without serum and in standard tissue flasks without mesenchymal cell feeder layers.
The culture medium used for phase 1 is designated complete MCDB 153 and consists of epidermal growth factor, insulin, bovine pituitary extract, ethanolamine, phosphoethanolamine, and hydrocortisone.
Phase 2 involves culturing of keratinocytes in Dulbecco's modified Eagle medium that contains serum, thereby facilitating cell stratification and differentiation.
B. A. Gilchrest et al. describe in "Attachment and growth of human keratinocytes in a serum-free environment" (Journal of Cellular Physiology, vol. 112, pp. 197-206, 1982) a culture medium for keratinocytes containing bovine brain extracts. In this document, there is no mention of wound healing properties.
However, these culture media (of Pittelkow et al. and of Gilchrest et al., see above) contain bovine pituitary extracts or bovine brain extracts, which implies:
that said media are not standardized; PA1 that confluency is reached only with difficulties; and PA1 that special substrates may be required.
Media containing no fibroblasts and no bovine organ extract have also been developed.
C. L. Marcello et al. have in their publication entitled "Stratification, specialization, and proliferation of primary keratinocyte cultures" (J. Cell. Biology, vol. 79, pp. 356-370, November 1978), described the culture of mouse keratinocytes in a medium containing Medium 199 and fetal calf serum. The culture temperature ranges between 32.degree.-33.degree. C. In this process, the seeding density is high (2.times.10.sup.5 cells/cm.sup.2) and confluence is reached in 4-6 weeks. The drawback is that this process results in low yields of keratinocyte sheets. In this document, there is no mention of wound healing properties.
Y. Kitano et al. in "Growth of human keratinocytes in a defined medium supplemented with growth factor of serum" (Dermatologica, vol. 180, pp. 236-239, 1990) have described that the cultures of keratinocytes were started by inoculating the keratinocytes suspended in Eagle's minimal essential medium supplemented with 20% fetal bovine serum, except for the experiments related to cell attachment. The basal medium consisted of equal volumes of Iscove's medium and Ham's F-12 medium supplemented with insulin, transferrin, ethanolamine, selenite, hydrocortisone and epidermal growth factor. The salting out of bovine serum was then performed in a conventional manner to fractionate bovine serum. In this document, there is no mention of wound healing properties. It is not clear from this article whether confluency is reached, which makes the cultured cells inappropriate for wound healing.
M. Eisinger et al. have described in "Human epidermal cell cultures: Growth and differentiation in the absence of dermal components or medium supplements" (Proc. Natl. Acad. Sci. USA, vol. 76, No. 10, pp. 5340-5344, October 1979), the human epidermal cell growth and differentiation in vitro, provided that the pH was 5.6-5.8, the seeding density was 2.5.times.10.sup.5 cells and the temperature was maintained at 35.degree.-37.degree. C.
The medium contained Eagle's minimal medium plus nonessential amino acids, 2 mM L-glutamine, hydrocortisone at 0.4 .mu.g/ml, 10% fetal bovine serum, penicillin, streptomycin and Fungizone. It takes 3 to 6 weeks before the confluent cultures (that can be used for preparation of cell extracts) are obtained. The drawbacks are the necessity of high seeding density and the slow growth of the cell cultures.
P. Hawley-Nelson et al. have described in "Optimized conditions for the growth of human epidermal cells in culture" (The Journal of Investigative Dermatology vol. 75, pp. 176-182, 1980) methods to optimize the growth of human keratinocytes in the following media:
CMRL 1066, Dulbecco's MEM with nonessential amino acids, Eagle's MEM with nonessential amino acids, Eagle's MEM with nonessential amino acids with D-valine substituted for L-valine, and BGJ.sub.b Fitton-Jackson modification. Two custom media were also tested: a modification of Eagle's MEM with 4-fold higher amino acids and vitamins (Fusening NE, Worst PKM: Mouse epidermal cell cultures. II. Isolation, characterization and cultivation of epidermal cells from perinatal mouse skin. Exp Cell Res. 93:443-457, 1975), and a modification of Waymouth's MB752/1 with added nonessential amino acids, putrescine, insulin, pyruvate, arginine, hydrocortisone and epidermal growth factor (Steele VE, Marchok AC, Nettesheim P., Transformation of tracheal epithelium exposed in vitro to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Int J. Cancer 20:234-238, 1977).
Primary epidermal cells grew to confluency within TGF-a2 weeks for plating inputs of 10.sup.5 /CM.sup.2.
Moreover, epidermal growth factor exposure resulted in the rapid appearance of a cell type which lacked the characteristic keratinocyte morphology and proliferated rapidly. Because the origin of these cells has not yet been determined, epidermal growth factor was not used further. In the above-mentioned article, there is no mention of wound healing properties.